Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide. Presently known virus isolates have been grouped into four different species which together form one genus within the family Flaviviridae.    I./II. Bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and type 2 (BVDV-2) cause bovine viral diarrhea (BVD) and mucosal disease (MD) in cattle (Baker, 1987; Moennig and Plagemann, 1992; Thiel et al., 1996). The division of BVDV into 2 species is based on significant differences at the level of genomic sequences (summarized in Heinz et al., 2000) which are also obvious from limited cross neutralizing antibody reactions (Ridpath et al. 1994).    III. Classical swine fever virus (CSFV), formerly named hog cholera virus, is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992; Thiel et al., 1996).    IV. Border disease virus (BDV) is typically found in sheep and causes border disease (BD). After intrauterine infection of lambs with BDV persistently infected lambs can be born that are weak and show different abnormalities among which the “hairy shaker” syndrome is best known (Moennig and Plagemann, 1992; Thiel et al., 1996).
Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5′ cap and 3′ poly(A) sequences. The viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and post-translational processing involving cellular and viral proteases. The viral proteins are arranged in the polyprotein in the order NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Lindenbach and Rice, 2001). Protein C (=core- or capsidprotein) and the glycoproteins Erns, E1, and E2 represent structural components of the pestivirus virion as demonstrated for CSFV (Thiel et al., 1991). This also holds true for BVDV. E2 and, to a lesser extent, Erns were found to be targets for antibody neutralization (Donis et al., 1988; Paton et al., 1992; van Rijn et al., 1993; Weiland et al., 1990, 1992). Erns lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus Erns and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993). Exchange of each of these residues against lysine in the Erns protein of a CSFV vaccine strain resulted in the destruction of RNase activity (Hulst et al., 1998). Introduction of these mutations into the genome of the CSFV vaccine strain did not influence viral viability or growth properties but led to a virus exhibiting a cytopathogenic phenotype (Hulst et al., 1998). Similarly, Meyers et al. showed that an RNase negative variant of the virulent CSFV strain Alfort/Tübingen was fully viable. However, the respective virus mutant showed no cytopathogenic phenotype (Meyers et al., 1999).
Npro represents the first protein encoded by the long open reading frame in the pestivirus RNA. Npro represents a nonstructural protein that has protease activity and cleaves itself of the nascent polyprotein (Stark et al., 1993; Wiskerchen et al., 1991) presumably already during translation. Npro is a cysteine protease (Rümenapf et al., 1998) that is not essential for virus replication (Tratschin et al., 1998). Recently, it was shown that Npro somehow interferes with the cellular antiviral defense so that it can be hypothesized to modulate the immune system within an infected host (Rüggli et al., 2003). Mayer and coworkers presented indications for an attenuation of CSFV in consequence of a deletion of the Npro gene (Mayer et al., 2004).
Present BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks (Oirschot et al., 1999). Vaccines against the classical BVDV-1 provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVDV-2 (Bolin et al., 1991; Ridpath et al., 1994). This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization (Tijssen et al., 1996). Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses (Ridpath et al., 1994).
Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used. Killed vaccines (inactivated whole virus) or subunit vaccines (conventionally purified or heterologously expressed viral proteins) are most often inferior to live vaccines in their efficacy to produce a full protective immune response even in the presence of adjuvants.
The structural basis of the attenuation of BVDV used as live vaccines is not known. These vaccines, although attenuated, are most often associated with safety problems. The vaccine viruses may cross the placenta of pregnant animals, e.g., cows, and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves. Therefore, they cannot be applied to breeding herds that contain pregnant cows. Pregnant cows have to be kept separate from vaccinated cattle to protect fetuses and must not be vaccinated themselves. Furthermore, revertants of attenuated live BVDV pose a serious threat to animals. For conventionally derived attenuated viruses wherein the attenuation is achieved by conventional multiple passaging, the molecular origin as well as the genetic stability of the attenuation remains unknown and reversion to the virulent wild-type is unpredictable.
Because of the importance of an effective and safe as well as detectable prophylaxis and treatment of pestiviral infections, there is a strong need for improved attenuated pestiviruses, such as BVDV, with a high potential for induction of immunity as well as a defined basis of attenuation which can also be distinguished from pathogenic pestiviruses, such as BVDV, as well as compositions and vaccines comprising the attenuated pestiviruses, such as BVDV.
Therefore, the technical problem underlying the present invention is to provide improved attenuated pestivirus, preferably an attenuated BVDV for use as live attenuated vaccines. Such improved attenuated pestivirus, preferably BVDV, should especially (i) not cross the placenta themselves, and (ii) induce an immunity that prevents viral transmission across the placenta and thereby prevents pregnancy problems like abortion of the fetus or birth of persistently infected host such calves in the case of BVDV infection.
All subsequent sequences show the deleted regions indicated with dashes (−), which are also numbered, whereas the sequences in the sequence listing attached hereto are continuously numbered without the deleted regions or amino acid codons.
SEQ ID NO: 1XIKE-A-cDNA sequenceSEQ ID NO: 2XIKE-A-NdN-cDNA sequenceSEQ ID NO: 3XIKE-B-cDNA sequenceSEQ ID NO: 4XIKE-B-NdN-cDNASEQ ID NO: 5XIKE-A amino acid sequenceSEQ ID NO: 6XIKE-A-NdN amino acid sequenceSEQ ID NO: 7XIKE-B amino acid sequenceSEQ ID NO: 8XIKE-B-NdN amino acid sequenceSEQ ID NO: 9XIKE-C-NdN amino acid sequenceSEQ ID NO: 10XIKE-C-NdN-cDNA sequenceSEQ ID NO: 11XIKE-C-cDNA sequenceSEQ ID NO: 12XIKE-C amino acid sequence